The present invention relates to the field of vaccinology and, in particular, to a multi-component vaccine comprising recombinant proteins from Haemophilus influenzae which is useful in protecting against disease caused by Haemophilus influenzae including otitis media.
Haemophilus influenzae is the cause of several serious human diseases such as meningitis, epiglottitis, septicemia and otitis media. There are six serotypes of H. influenzae, designated a to f, that are identified by their capsular polysaccharide. H. influenzae type b (Hib) was a major cause of bacterial meningitis until the introduction of several Hib conjugate vaccines in the 1980""s (ref. 1, throughout this application, various references are referred to in parenthesis to more fully describe the state of the art to which this invention pertains Full bibliographic information for each citation is found at the end of the specification immediately preceding the claims. The disclosures of these references are hereby incorporated by reference into the present disclosure). Vaccines based upon H. influenzae type b capsular polysaccharide conjugated to diphtheria toxoid (ref. 2), tetanus toxoid (ref. 3 and U.S. Pat. No. 4,496,538), or Neisseria meningitidis outer membrane protein (ref. 4) have been effective in reducing H. influenzae type b-induced meningitis. The other serotypes of H. influenzae are associated with invasive disease at low frequencies, although there appears to be an increase in the incidence in disease caused by these strains as the incidence of Hib disease declines (refs. 5 and 6). Non-encapsulated or nontypeable H. influenzae (NTHi) are also responsible for a wide range of human diseases including otitis media, epiglottitis, pneumonia and tracheobronchitis. The incidence of NTHi-induced disease has not been affected by the introduction of the Hib vaccines (ref. 7).
Otitis media is the most common illness of early childhood, with 60 to 70% of all children, of less than 2 years of age, experiencing between one and three ear infections (ref. 8). Chronic otitis media is responsible for hearing, speech and cognitive impairments in children. H. influenzae infections account for about 30% of the cases of acute otitis media and about 60% of chronic otitis media. In the United States alone, treatment of otitis media costs between 1 and 2 billion dollars per year for antibiotics and surgical procedures, such as tonsillectomies, adenoidectomies and insertion of tympanostomy tubes. It is estimated that an additional $30 billion is spent per annum on adjunct therapies, such as speech therapy and special education classes. Furthermore, many of the causative organisms of otitis media are becoming resistant to antibiotic treatment. An effective prophylactic vaccine against otitis media is thus desirable.
During natural infection, surface-exposed outer membrane proteins that stimulate an antibody response are potentially important targets for bactericidal and/or protective antibodies and therefore potential vaccine candidates. Barenkamp and Bodor (ref. 9) demonstrated that convalescent sera from children suffering from otitis media due to NTHi, contained antibodies to high molecular weight (HMW) proteins. About 70 to 75% of NTHi strains express the HMW proteins and most of these strains contain two gene clusters termed hmw1ABC and hmw2ABC (refs. 10, 11). The HMWA proteins have been demonstrated to be adhesins mediating attachment to human epithelial cells (ref. 12). Immunization with a mixture of native HMW1A and HMW2A proteins resulted in partial protection in the chinchilla intrabulla challenge model of otitis media (ref. 13)
U.S. Pat. No. 5,603,938 (Barenkamp), assigned to St. Louis University and Washington University and the disclosure of which is incorporated herein by reference, describes the cloning, expression and sequencing of the genes encoding the HMW1 and HMW2 proteins from strain 12 of non-typeable Haemophilus influenzae. The HMW proteins are a family of proteins from non-typeable Haemophilus influenzae of molecular weight of about 120 to 125 kDa which are absent from encapsulated strains of Haemophilus influenzae. 
The production of native HMW proteins from H. influenzae strains is very low and a method for producing protective recombinant HMW (rHMW) proteins has been described in copending U.S. patent application Ser. No. 09/167,568 filed Oct. 7, 1998, assigned to the assignee hereof and the disclosure of which is incorporated herein by reference. A chinchilla nasopharyngeal colonization model has been developed specifically to demonstrate vaccine efficacy of adhesins (ref. 14) and the rHMW proteins are protective in this model as described in the aforementioned copending U.S. patent application Ser. No. 09/167,568. The rHMW1A and rHMW2A proteins were shown to afford equivalent protection and the rHMW1A protein was chosen for further vaccine studies. In this application, rHMW refers to recombinant HMW1A from NTHi strain 12, although other corresponding recombinant HMW1A proteins from other NTHi strains and corresponding HMW2A proteins from NTHi strains may be employed. The corresponding naturally-occurring proteins may be employed.
A second family of high molecular weight adhesion proteins has been identified in about 25% of NTHI and in encapsulated H. influenzae strains (refs. 15, 16, 17). The NTHi member of this second family is termed Haemophilus influenzae adhesin or Hia and the homologous protein found in encapsulated strains is termed Haemophilus influenzae surface fibril protein or Hsf.
U.S. Pat. No. 5,646,259 (St. Geme, III et al), assigned to St. Louis University and Washington University, and the disclosure of which is incorporated herein by reference, describes the cloning, expression and sequences of genes encoding the Hia and Hsf proteins, which have limited homology to the HMW1 and HMW2 proteins of U.S. Pat. No. 5,603,938.
The hia gene was originally cloned from an expression library using convalescent sera from an otitis media patient, which indicates that it is an important immunogen during disease. The prototype Hia and Hsf proteins demonstrate about 82% sequence similarity, although the Hsf protein is considerably larger. The proteins are comprised of conserved amino and carboxy termini and several repeat motifs, with Hsf containing more repeat sequences than Hia.
United States patent application Ser. No. 09/268,347 filed Mar. 26, 1999, assigned to the assignee hereof and the disclosure of which is incorporated herein by reference, describes the production of full-length and N-terminal truncated versions of the Hia protein (rHia) in E. coli. These recombinant proteins have been demonstrated to protect against bacteremia caused by H. influenzae type a and type b organisms, and to confer partial protection against nasopharyngeal colonization by non-typeable H. influenzae. In this application, rHia refers to V38 rHia from NTHi strain 11, although other recombinant full-length and N-terminal truncated Hia proteins from other NTHi strains may be employed. The corresponding naturally-occurring proteins may be employed. The corresponding naturally-occurring proteins may be employed.
When under environmental stress, such as high temperature, organisms overproduce stress response or heat shock proteins (hsps). Bacterial hsps have been shown to be important immunogens, stimulating both B cells and T cells (ref. 18). The bacterial HtrA or DegP heat shock proteins are expressed under conditions of stress and the H. influenzae HtrA or Hin47 protein has been shown to be a partially protective antigen in the intrabulla challenge model of otitis media (ref. 19). The HtrA proteins are serine proteases and their proteolytic activity makes them unstable. In addition, as components of a multicomponent vaccine, the wild-type HtrA protein will degrade mixed antigens. The site-directed mutagenesis of the H. influenzae htrA gene (termed hin47) and the properties of the mutants have been fully described in U.S. Pat. No. 5,506,159 (Loosmore et al), assigned to the assignee hereof and the disclosure of which is incorporated herein by reference.
U.S. Pat. No. 5,506,139 (Loosmore et al) describes the preparation of analogs of Haemophilus influenzae Hin47 protein which have a decreased protease activity which is less than about 10% of that of the natural Hin47 protein and which preferably have substantially the same immunogenic properties as natural Hin47 protein. The patent also describes the isolation, purification and characterization of nucleic acid molecules encoding the Hin47 analogs. The natural Hin47 protein is immunologically conserved among non-typeable and type b isolates of H. influenzae. The amino acid sequence of the natural Hin47 protein and the nucleotide sequence of the encoding hin47 gene are described in WO 94/00149 published Jan. 6, 1994 and incorporated herein by reference.
The Hin47 analogs of U.S. Pat. No. 5,506,139 are prepared by deleting or replacing by a different amino acid at least one amino acid of the natural Hin47 contributing to protease activity or by inserting at least one amino acid into the natural Hin47 protein, as specifically described therein. The at least one deleted or replaced amino acid may be selected from amino acids 195 to 201 of Hin47 and specifically may be Serine-197, which may be deleted or replaced by alanine. In addition, the at least one deleted or replaced amino acid may be His-91 and may be deleted or replaced by alanine, lysine or arginine. Furthermore the at least one deleted or replaced amino acid may be Asp-121 and may be deleted or replaced by alanine.
In U.S. patent application Ser. No. 08/487,167 filed Jun. 7, 1995, assigned to the assignee hereof and the disclosure of which is incorporated herein by reference (now U.S. Pat. No. 5,869,302), there are described multiple mutations effected at different amino acids of the natural Hin47 protein to provide the non-proteolytic Hin47 analog.
In the present invention, the mutation of histidine 91 to alanine (sometimes termed herein xe2x80x9cH91Axe2x80x9d) is employed as illustration of the mutant Hin47 protein, although other Hin47 mutants with reduced protease activity as described in the aforementioned patent and application may be used.
The non-proteolytic HtrA analogue H91A Hin47, has been shown to be a protective antigen against bacteremia caused by H. influenzae type b and against otitis media caused by non-typeable H. influenzae (ref. 19). HtrA was found in all strains examined, including all encapsulated strains of H. influenzae. 
The main goal of a prophylactic vaccine against otitis media is to prevent the establishment of nasopharyngeal colonization by including adhesins as immunogens. The H. influenzae HMW and Hia proteins are adhesins that have been shown to prevent colonization. However, since there may be a small percentage of H. influenzae strains that do not contain the hmw or hia genes and a wide spectrum of disease protection is desired, the H91A Hin47 antigen has been added to provide protection against such strains, although any other non-proteolytic analogs of Hin47 may be employed. The present invention provides for a multi-component vaccine to protect against colonization and disease caused by encapsulated or non-encapsulated H. influenzae organisms.
It would be desirable to provide efficacious combination vaccines comprising H. influenzae components containing selected relative amounts of selected antigens.
The present invention is directed towards the provision of a multi-component vaccine comprising at least three antigens, to protect against disease caused by infection with Haemophilus influenzae, including otitis media.
In accordance with one aspect of the present invention, there is provided an immunogenic composition for conferring protection in a host against disease caused by infection by Haemophilus influenzae, including otitis media, comprising at least three different antigens of Haemophilus influenzae, at least two of which different antigens are adhesins.
One of the antigens which is an adhesin may be a high molecular weight protein (HMW) of a non-typeable strain of Haemophilus, particularly an HMW1 or HMW2 protein of the non-typeable strain, which may be produced recombinantly.
The other of the antigens which is an adhesin is a Haemophilus influenzae adhesin (Hia) protein of a non-typeable strain of Haemophilus influenzae or a Haemophilus influenzae surface fibril (Hsf) protein of a typeable strain of Haemophilus influenzae. 
The antigen of Haemophilus influenzae which is not an adhesin may be a non-proteolytic heat shock protein of a strain of Haemophilus influenzae. The non-proteolytic heat shock protein of a strain of Haemophilus influenzae may be an analog of Haemophilus influenzae Hin47 protein having a decreased protease activity which is less than about 10% of that of the natural Hin47 protein.
In accordance with a preferred embodiment of the aspect of the invention, there is provided an immunogenic composition for conferring protection in a host against disease caused by Haemophilus influenzae, including otitis media, which comprises:
(a) an analog of Haemophilus influenzae Hin47 protein having a decreased protease activity which is less than about 10% of natural Hin47 protein,
(b) a Haemophilus influenzae adhesin (Hia) protein of a non-typeable strain of Haemophilus influenzae, and
(c) a high molecular weight (HMW) protein of a strain of non-typeable Haemophilus influenzae. 
The analog of Hin47 protein may be one in which at least one amino acid of the natural Hin47 protein contributing to protease activity has been deleted or replaced by a different amino acid and which has substantially the same immunogenic properties as natural Hin47 protein.
Such at least one amino acid may be selected from the group consisting of amino acids 91, 121 and 195 to 207 of natural Hin47 protein. Specific mutants which may be used including serine-197 replaced by alanine, Histidine-91 replaced by alanine, lysine or arginine and Asp-121 replaced by alanine.
The HMW protein of the non-typeable strain of Haemophilus influenzae may be a HMW1 or HMW2 protein and may be recombinantly produced. The HMW1 and HMW2 proteins are derived from the respective strains of non-typeable Haemophilus influenzae and possess respective molecular weights as set forth in the following Table I:
The Hia protein may be produced recombinantly and may comprise the N-terminal truncated V38 rHia protein.
The immunogenic composition of the invention may be further formulated with an adjuvant. Such adjuvant for use in the present invention may include (but not limited to) aluminum phosphate, aluminum hydroxide, QS21, Quil A, derivatives and components thereof, ISCOM matrix, calcium phosphate, calcium hydroxide, zinc hydroxide, a glycolipid analog, an octadecyl ester of an amino acid, a muramyl dipeptide, polyphosphazene, ISCOPREP, DC-chol, DDBA and a lipoprotein and other adjuvants. Advantageous combinations of adjuvants are described in copending U.S. patent applications Ser. No. 08/261,194 filed Jun. 16, 1994 and 08/483,856 filed Jun. 7, 1995, assigned to the assignee hereof and the disclosures of which are incorporated herein by reference (WO 95/34308, published Nov. 21, 1995). The adjuvant preferably may comprise aluminum phosphate or aluminum hydroxide (collectively known as alum).
The components of the immunogenic composition may be present in appropriate quantities to provide the desired immune response. The components may be formulated as a vaccine for in vivo administration to the host. The vaccine composition may contain:
(a) about 25 to 100 xcexcg of the Hin47 protien,
(b) about 25 to 100 xcexcg of the Hia protien, and
(c) about 25 to 100 xcexcg of the HMW protien.
The immunogenic compositions may be formulated with other antigenic components to provide a multivalent vaccine in which the additional antigenic component(s) confer protection against disease caused by another pathogen(s). Such additional antigens should be such that and be present in quantities that the immunogenicity of the individual components of the resulting vaccine is not impaired by other individual components of the composition. Such additional antigens preferably are purified antigens in defined quantities to produce a component vaccine.
Such additional antigens may be those traditionally found in multivalent protective vaccines, such as diphtheria toxoid, tetanus toxoid and pertussis antigens, including pertussis toxoid, filamentous hemagglutinin, pertactin and/or agglutinogens.
The resulting multivalent vaccine also may contain non-virulent poliovirus, such as inactivated poliovirus, which may be type 1, type 2 and/or type 3 poliovirus. The multi-component vaccine further may comprise a conjugate of a tetanus or diphtheria toxoid and a capsular polysaccharide of Haemophilus influenzae, preferably PRP-T.
The invention extends to a method of immunizing a host against disease caused by infection by Haemophilus influenzae, including otitis media, which comprises administering to the host an immunoeffective amount of the immunogenic composition provided herein.
Advantages of the present invention include a multi-component vaccine that can confer protection against encapsulated and non-encapsulated Haemophilus influenzae diseases in a safe and efficacious manner.